Cyanobacteria are considered to be the precursor of plant chloroplasts. Cyanobacteria possess all beneficial features of prokaryotes like ease of handling, rapid growth under defined conditions, availability of replica plating techniques, easy genetic manipulation by mutagenesis or transformation and availability of established mutants. Cyanobacterial metabolism also share important features with higher plant metabolism such as oxygenic photosynthesis by two photosystems and autotrophy with respect to reduced nitrogen, sulfur and carbon dioxide. Therefore, efficacy of compounds in cyanobacteria can be indicative of similar performance in higher plants.
The photosynthetic membranes of cyanobacteria, plants and algae contain essential pigments called carotenoids, which function in protecting chlorophyll against photo oxidative damage by singlet oxygen, as well as acting as accessory pigments in photosynthetic light harvesting. These carotenoids are also precursors of vitamin A in human and abscissic acid in plants. The first committed step in carotenoids biosynthesis is the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to yield the colorless phytoene. Desaturation of phytoene through the insertion of four double bonds gives rise to lycopene, and further cyclization reactions lead to the generation of β-carotene. Phytoene desaturase(pds) mediates the first two steps of desaturation of phytoene, disruption of which results in an observable bleaching symptom. As such, a number of commercial herbicides directed at inhibiting this enzyme have been developed, e.g. norflurazon, fluridone, and fluorochloridone.
In addition, as ancestral precursors to chloroplasts, cyanobacterial genes share features common to chloroplast genes. Gene elements, such as promoters, ribosome binding sites, etc. are similar and can be cross-functional between chloroplast and cyanobacteria. Therefore cyanobacterial genes make ideal candidates for plastome targeted transformation, and in particular chloroplast transformation.
There are a number of references in the literature to screening methods and assays utilizing cyanobacteria. These include methods using cyanobacteria for the screening of compounds to identify inhibitors of specific metabolic pathways and identification of novel herbicidal modes of action. [Hirschberg et al, 1996] describes an Erwinia gene transformed into host cells selected of cyanobacteria, specifically Synechococcus PCC 7942 and Synechocystis PCC 6803, which was used as a screen for beta-carotene biosynthesis and for mutants resistant to herbicides specifically bleaching herbicides of the trialkylamine family. The screening for bleaching activity is described by [Sandmann et al, 1991] as a means to discover new herbicides with different core structures which inhibit phytoene desaturase (pds), a membrane bound enzyme in the carotenogenic pathway catalyzing the hydrogen abstraction step at the first C40 precursor of beta-carotene. [Windhoevel et al, 1994] describes a screen involving genes coding for pds of the non-photosynthetic bacterium Erwinia uredovora introduced into the cyanobacterium Synechococcus as a convenient experimental model for higher plant transformation and resistance to herbicides. The functionality of the heterologously expressed phytoene desaturase in the transformants was demonstrated in assays. Other references such as [Babczinski et al, 1995] identify new herbicide class inhibiting pds based on a screen utilizing the unicellular cyanobacteria Anacystis. [Chamowitz et al, 1993] described a cell-free carotegenic assay to identify herbicide resistant algal pds mutants. Inhibition of carotenoid biosynthesis by herbicidal m-phenoxybenzamide derivatives was investigated in a cell-free in vitro assay using the cyanobacteria Aphanocapsa by [Clarke et al, 1985], and subsequently by [Kowalczyk-Schroeder et al, 1992]. [Sandmann et al, 1991], describes a non-radioactive cell-free assay to quantitatively determine inhibition of plant-type pds by bleaching herbicides. They further developed a cyanobacterial pds assay system, a mode of action assay utilizing the cyanobacteria Anacystis, and assays using algal cells. The present invention, however, differs by identifying improvements to the current screening methods and assays, and uses these improvements to identify novel nucleic acid fragments having herbicide resistance mutations in the pds gene.
The prokaryotic acetolactate synthase (ahas) enzyme exists as two distinct, but physically associated, protein subunits. In prokaryotes, the two polypeptides, a “large subunit” and a “small subunit” are expressed from separate genes. Three major ahas enzymes, designated I, II, III, all having large and small subunits have been identified in enteric bacteria. In prokaryotes, the ahas enzyme has been shown to be a regulatory enzyme in the branched amino acid biosynthetic pathway [Miflin et al, 1971], and only the large subunit has been observed as having catalytic activity. From studies of ahas enzymes from microbial systems, two roles have been described for the small subunit: 1) the small subunit is involved in the allosteric feedback inhibition of the catalytic large subunit when in the presence of isoleucine, leucine or valine or combinations thereof, and 2) the small subunit enhances the activity of the large subunit in the absence of isoleucine, leucine or valine. The small subunit has also been shown to increase the stability of the active conformation of the large subunit. The expression of the small subunit can also increase the expression of the large subunit as seen for AHAS I from E. coli [Weinstock et al., 1992].
The ahas large subunit protein has been identified in plants, and has also been isolated and used to transform plants. An ahas mutant allele isotype of the ahas III large subunit protein, having the tryptophan at position 557 replaced with leucine has been found in a Brassica napus cell line [Hattori et al., 1995]. The mutant protein product of this gene confers sulfonylurea, imidazolinone and triazolopyridine resistance to the cell line. This mutant allele, when expressed in transgenic plants, also confers resistance to these herbicides.
Until recently, there was no direct evidence that a small subunit protein of ahas exisited in eukaryotic organisms. Recently, other groups, through the use of Expressed Sequence Tags (ESTs), have identified sequences homologous to the microbial ahas small subunit genes in an eukaryote, the plant Arabidopsis. These groups showed that a randomly isolated Arabidopsis cDNA sequence had sequence homology with the ahas small subunit sequences from microbial systems. Since then, ESTs from small subunit genes have been described from other eukaryotes such as yeast and red algae. [Duggleby et al, 1997] describes three EST sequences, two from Arabidopsis and one from rice, that have homology to known prokaryotic small subunit cDNA sequence from P. purpurea. 
Several references to ahas screens and assays utilizing cyanobacteria exist in the prior art. [Powell et al, 1990], reported on the role of cyanobacteria for herbicide screening but no mention was made of the ahas “small subunit” identified in our invention. They reported that our understanding of the mode of action of certain herbicides which inhibit photosynthesis has been facilitated by studies with cyanobacteria. In the case of sulfonylurea herbicides which inhibit branched chain amino acid biosynthesis, the resistance shown by a cyanobacterium is due to an insensitive acetolactate synthase enzyme. These studies are not consistent with the results reported by Freiburg et al. discussed below, in which the cyanobacterial gene is sensitive. If other insensitive target enzymes were to be found, cyanobacteria could be useful sources of genes for the cloning of herbicide resistance into higher plants. They presented data showing high levels of resistance of certain cyanobacteria to glyphosate, an inhibitor of aromatic amino acid biosythesis. [Dunahay et al, 1997], discloses a method to transform chlorophyll containing algae, which includes introducing a recombinant molecule comprising a nucleic acid molecule encoding a dominant selectable marker operatively linked to an algal regulatory control sequence into the chlorophyll C-containing algae. However, unlike our invention, the mutant ahas was introduced into algae, not cyanophycae, to detect inhibitors.
WO 98/06862 (Calgene) discloses plants transformed with the Erwinia phytoene desaturase gene for altered carotenoid levels and fatty acid. JP 6,343,476 (Kirin Brewery) describes the production of bleaching herbicide-resistant plants by transformation with the Erwinia pds gene. WO 98/06862 (Zeneca) discloses transgenic plants resistant to many classes of herbicides but the source of the genes, whether pds or ahas or from Synechocystis is unspecified. Also, U.S. Pat. No. 5,378,824 (Dupont) and U.S. Pat. No. 5,661,017 (Dunahay et al.) both report the transformation of a plant ahas gene, not a Synechocystis gene, into a number of phyla and classes including algae.
Freiburg et al, 1990, reported on herbicide resistant Synechococcus ahas gene expressed in E. coli. The report describes the isolation and molecular characterization of acetolactate synthase genes from the sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant, which specifies the enzyme resistant to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an E. coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The resistant phenotype is a consequence of proline to serine substitution in residue 114 of the deduced amino acid sequence. Functional expression of the mutant gene in Synechococcus and in E. coli confirmed that this amino acid sequence is responsible for the resistance. [Linden et al, 1990], reported cyanobacteria Synechococcus PCC 7942 mutants selected against the bleaching herbicide norflurazon. One strain exhibited cross-resistance against another bleaching herbicide fluorochloridone, but the other three strains did not show cross-resistance against other phytoene desaturase (pds) inhibitors. [Sandman et al, 1991] reported on mutants from Synechococcus PCC 7942, which were selected for tolerance to various bleaching herbicides. A mutant NFZ4 established a high degree of cross-resistance to both norflurazon and fluorochloridone, but not to fluridone. [Chamowitz et al, 1991] cloned and sequenced a pds gene from the cyanobacteria Synechococcus PCC 7942, also resistant to the bleaching herbicide norflurazon. The identified mutant is a ValGly change at position 403 in the Synechococcus but not Synechocystis pds protein. [Sandmann et al, 1998] reported bacterial and fungal pds as a target for bleaching herbicides, and discussed the identification of cyanobacterial mutants with resistance to specific compounds and their cross-resistance to other bleaching herbicides.
Cyanobacteria Synechocystis was originally described in Vioque et al, 1992. A spontaneous mutant, strain AV4, which is resistant to norflurazon, was isolated from Synechocystis PC 6803. DNA isolated from the mutant AV4 can transform wild-type cells to norflurazon resistance with high frequency. Sequence analysis of the clone identified an open reading frame that is highly homologous to the previously sequenced pds genes from Synechococcus and soybean. In both cyanobacteria and plants the pds gene is highly conserved: the Synechocystis PCC 6803 pds gene is 82% and 61% identical to the Synechococcus PCC 7942 and the soybean pds genes respectively. [Sandmann et al, 1994] identified three distinct Synechocystis mutants selected against norflurazon, and showed modification of the same amino-acid of phytoene desaturase into three different ones. In all cases, the same amino-acid Arg195 was modified either into Cys, Pro or Ser. The degree of resistance was highest when Arg was changed into Ser.
While the literature has several references to pds herbicide resistant transgenic plants, our intervention exemplifies improvements to current cyanobacteria screening methods. Our improvement has identified novel nucleic acid fragments from Synechocystis PCC 6803. The mutant pds (phytoene desaturase) gene and ahas large and small subunits are useful in the identification of novel pds and ahas inhibitors and, in plant transformations for conferring resistance and cross-resistance to certain bleaching herbicides and AHAS-inhibiting herbicides.